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1.
Neurobiol Dis ; 184: 106210, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37352984

RESUMO

The progressive neurodegenerative disease amyotrophic lateral sclerosis (ALS) is caused by a decline in motor neuron function, resulting in worsened motor impairments, malnutrition, respiratory failure and mortality, and there is a lack of effective clinical treatments. The exact mechanism of motor neuronal degeneration remains unclear. Previously, we reported that ferroptosis, which is characterized by the accumulation of lipid peroxide and glutathione depletion in an iron-dependent manner, contributed to motor neuronal death in ALS cell models with the hSOD1G93A (human Cu/Zn-superoxide dismutase) gene mutation. In this study, we further explored the role of ferroptosis in motor neurons and its regulation in mutant hSOD1G93A cell and mouse models. Our results showed that ferroptosis was activated in hSOD1G93A NSC-34 cells and mouse models, which was accompanied by decreased nuclear retention of nuclear factor erythroid 2-related factor 2 (NRF2) and downregulation of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) levels. Moreover, RTA-408, an NRF2 activator, inhibited ferroptosis in hSOD1G93A NSC-34 cells by upregulating the protein expression of SLC7A11 and GPX4. Moreover, hSOD1G93A mice treated with RTA-408 showed obvious improvements in body weight and motor function. Our study demonstrated that ferroptosis contributed to the toxicity of motor neurons and that activating NRF2 could alleviate neuronal degeneration in ALS with the hSOD1G93A mutation.


Assuntos
Esclerose Lateral Amiotrófica , Ferroptose , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Neurônios Motores/metabolismo , Mutação/genética , Doenças Neurodegenerativas/metabolismo , Neuroproteção , Fator 2 Relacionado a NF-E2/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
2.
Brain Behav Immun ; 110: 60-79, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36754245

RESUMO

BACKGROUND: Chronic alcohol exposure (CAE) during late adolescence increases the risk of anxiety development. Alcohol-induced prefrontal cortex (PFC) microglial activation, characterized by morphological changes and increased associations with neurons, plays a critical role in the pathogenesis of anxiety. Alcohol exposure increases NLRP3 inflammasome expression, increasing cytokine secretion by activated microglia. Cannabinoid type 2 receptor (CB2R), an essential receptor of the endocannabinoid system, regulates microglial activation and neuroinflammatory reactions. We aimed to investigate the role of CB2R activation in ameliorating late adolescent CAE-induced anxiety-like behaviors and microglial activation in C57BL/6J mice. METHODS: Six-week-old C57BL/6J mice were acclimated for 7 days and then were administered alcohol by gavage (4 g/kg, 25 % w/v) for 28 days. The mice were intraperitoneally injected with the specific CB2R agonist AM1241 1 h before alcohol treatment. Anxiety-like behaviors during withdrawal were assessed by open field test and elevated plus maze test 24 h after the last alcohol administration. Microglial activation, microglia-neuron interactions, and CB2R and NLRP3 inflammasome-related molecule expression in the PFC were measured using immunofluorescence, immunohistochemical, qPCR, and Western blotting assays. Microglial morphology was evaluated by Sholl analysis and the cell body-to-total cell size index. Additionally, N9 microglia were activated by LPS in vitro, and the effects of AM1241 on NLRP3 and N9 microglial activation were investigated. RESULTS: After CAE, mice exhibited severe anxiety-like behaviors during withdrawal. CAE induced obvious microglia-neuron associations, and increased expression of microglial activation markers, CB2R, and NLRP3 inflammasome-related molecules in the PFC. Microglia also showed marked filament retraction and reduction and cell body enlargement after CAE. AM1241 treatment ameliorated anxiety-like behaviors in CAE model mice, and it prevented microglial morphological changes, reduced microglial activation marker expression, and suppressed the microglial NLRP3 inflammasome activation and proinflammatory cytokine secretion induced by CAE. AM1241 suppressed the LPS-induced increase in NLRP3 inflammasome-related molecules, IL-1ß release, and M1 phenotype markers (iNOS and CD86) in N9 cell, which was reversed by CB2R antagonist treatment. CONCLUSIONS: CAE caused anxiety-like behaviors in late adolescent mice at least partly by inducing microglial activation and increasing microglia-neuron associations in the PFC. CB2R activation ameliorated these effects by preventing morphological changes and suppressing NLRP3 inflammasome activation in PFC microglia.


Assuntos
Ansiedade , Etanol , Inflamassomos , Microglia , Córtex Pré-Frontal , Receptor CB2 de Canabinoide , Animais , Camundongos , Consumo de Bebidas Alcoólicas/efeitos adversos , Ansiedade/etiologia , Ansiedade/metabolismo , Canabinoides/farmacologia , Citocinas/metabolismo , Etanol/efeitos adversos , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Córtex Pré-Frontal/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Receptores de Canabinoides/metabolismo
3.
Am J Drug Alcohol Abuse ; 48(5): 562-572, 2022 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-35838410

RESUMO

B a ckground: Circular RNAs (circRNAs) have been crucially implicated in various diseases, however, their involvement in chronic intermittent ethanol (CIE) exposure remains unclear.O bjective: The present study was conducted to evaluate the circular RNA expression alteration in brain samples and to identify the molecular mechanisms underlying chronic intermittent ethanol exposure.M ethods: Male C57BL/6J mice (10 for each group) were given 4 weeks of chronic intermittent ethanol exposure. Whole brain samples were collected for high-throughput sequencing and circRNA bioinformatic analysis. Real-time quantitative PCR (RI-qPCR) and agarose electrophoresis were used to validate the differentially expressed circRNAs. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis were performed. A p level < 0.05 was considered statistically significant.R esults: Compared with the control group and baseline values, the CIE group showed a significant increase in ethanol intake. High-throughput sequencing revealed 399 significantly different circRNAs in CIE mice, including 150 up-regulated circRNAs and 249 down-regulated circRNAs. GO analysis showed that the most significantly enriched term for biological process, cellular component, and molecular function were GO:0050885, GO:0016020 and GO:0005515, respectively. The most enriched pathways in KEGG analysis were GABAergic synapse (mmu04727), followed by retrograde endocannabinoid (eCB) signaling (mmu04723) and morphine addiction (mmu05032). Among the circRNAs, RT-qPCR confirmed 14 upregulated and 13 downregulated circRNAs in the brain tissues with 9 upregulated and 10 downregulated circRNAs being observed in blood samples.C onclusions: Our study suggests that chronic ethanol exposure upregulates or downregulates circRNAs in the brain, which, in turn, could alter neurotransmitter release and signal transduction.


Assuntos
Etanol , RNA Circular , Animais , Regulação para Baixo , Endocanabinoides , Perfilação da Expressão Gênica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurotransmissores , RNA Circular/genética , Sefarose , Regulação para Cima
4.
Int J Neurosci ; 130(9): 857-864, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31928267

RESUMO

Background and purpose: The optimal treatment for acute ischemic stroke with mild neurologic deficits is unclear. We aimed to compare the efficacy and safety of alteplase versus dual-antiplatelet therapy in acute minor stroke.Methods: We performed a retrospective cohort study of patients with minor ischemic stroke and National Institutes of Health Stroke Scale scores ≤5 presenting within 24 h from last seen normal. Patients were divided into intravenous alteplase or dual-antiplatelet therapy group. The primary outcome was a modified Rankin Scale (mRS) score of 0 or 1 at 90 days. Secondary outcomes included mRS score at 7 days, and composite outcome of vascular events within 90 days. The safety outcome was any intracranial hemorrhage (ICH) according to the ECASS II criteria. Clinical outcomes were compared using a multivariable logistic regression after adjusting for confounding factors. We then performed the propensity score matching as a sensitivity analysis.Results: Two hundred twenty-eight patients met the eligibility criteria were included for analysis between January 2015 and September 2018. In the aspirin-clopidogrel group, 109 patients (91.6%) achieved a favorable functional outcome at 3-month versus 85(78.0%) in the alteplase group (OR 4.463, 95%CI 1.708-11.662, p = .002). The difference of the composite outcome of vascular events were not statistical significance between the two groups (p > .05). Asymptomatic ICH occurred in 0.8% patients who received aspirin-clopidogrel, as compared with 3.7% patients in alteplase group (p = .030).Conclusions: Patients treated with dual-antiplatelet therapy with acute minor ischemic stroke had greater functional outcome at 3 months compared with patients who received alteplase therapy.Classification of evidence: This study provides Class IV evidence that dual-antiplatelet therapy is superior to alteplase for achieving a better functional outcome and does not increase the risk of hemorrhage in acute minor ischemic stroke.


Assuntos
Aspirina/farmacologia , Clopidogrel/farmacologia , Fibrinolíticos/farmacologia , Hemorragias Intracranianas/induzido quimicamente , AVC Isquêmico/tratamento farmacológico , Avaliação de Resultados em Cuidados de Saúde , Inibidores da Agregação Plaquetária/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Idoso , Aspirina/efeitos adversos , Clopidogrel/efeitos adversos , Quimioterapia Combinada , Feminino , Fibrinolíticos/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/efeitos adversos , Estudos Retrospectivos , Índice de Gravidade de Doença , Ativador de Plasminogênio Tecidual/efeitos adversos
5.
Brain Behav Immun ; 82: 63-75, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31376498

RESUMO

Chronic excessive drinking leads to a wide spectrum of neurological disorders, including cognitive deficits, such as learning and memory impairment. However, the neurobiological mechanisms underlying these deleterious changes are still poorly understood. We conducted a comprehensive study to investigate the role and mechanism of autophagy in alcohol-induced memory impairment. To establish an ethanol-induced memory impairment mouse model, we allowed C57BL/6J mice intermittent access to 20% ethanol (four-bottle choice) to escalate ethanol drinking levels. Memory impairment was confirmed by a Morris water maze test. We found that mice exposed to EtOH (ethanol) and EtOH combined with the autophagy inhibitor 3-methyladenine (3-MA) showed high alcohol intake and blood alcohol concentration. We confirmed that the EtOH group exhibited notable memory impairment. Inhibition of autophagy by 3-MA worsened ethanol-induced memory impairment. Ethanol induced autophagy in the hippocampus of mice as indicated by western blotting, electron microscopy, RT-qPCR, and fluorescence confocal microscopy. We determined that the mTOR/BECN1 (S14) pathway is involved in ethanol-induced autophagy in vivo. Further, ethanol-induced autophagy suppressed the NLRP3 inflammatory and apoptosis pathways in the hippocampus in mice and in vitro. These findings suggest that autophagy activation in hippocampal cells alleviates ethanol-induced memory impairment in association with anti-apoptotic and anti-inflammatory pathways.


Assuntos
Autofagia/efeitos dos fármacos , Etanol/efeitos adversos , Memória/efeitos dos fármacos , Memória/fisiologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Concentração Alcoólica no Sangue , Morte Celular/efeitos dos fármacos , Disfunção Cognitiva/metabolismo , Etanol/farmacologia , Hipocampo/metabolismo , Masculino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia , Neuroimunomodulação , Serina-Treonina Quinases TOR/metabolismo
6.
J Neuroinflammation ; 14(1): 220, 2017 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-29132376

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) induces tumor immunosuppression through interacting with tumor-infiltrating microglia or macrophages (TAMs) with an unclear pathogenesis. Enhancer of zeste homolog 2 (EZH2) is abundant in GBM samples and cell lines and is involved in GBM proliferation, cell cycle, and invasion, whereas its association with innate immune response is not yet reported. Herein, the aim of this study was to investigate the role of EZH2 in GBM immune. METHODS: Co-culturing models of human/murine GBM cells with PBMC-derived macrophages/primary microglia were employed. EZH2 mRNAs and function were suppressed by siEZH2 and DZNep. Real-time PCR and flow cytometry were used to determine levels of microglia/macrophages markers. The fluorescence-labeled latex beads and flow cytometry were utilized to evaluate phagocytic abilities of microglia. CCK8 assay was performed to assess microglia proliferation. RESULTS: EZH2 inhibition led to significant reduction of TGFß1-3 and IL10 and elevation of IL1ß and IL6 in human and murine GBM cells. More importantly, EZH2 suppression in GBM cells resulted in significant increase of M1 markers (TNFα and iNOS) and decrease of a pool of M2 markers in murine microglia. The proportion of CD206+ cells was decreased in PBMC-derived macrophages as co-incubated with EZH2-inhibited GBM cells. Functional researches showed that phagocytic capacities of microglia were significantly ameliorated after EZH2 inhibition in co-culturing GBM cells and microglia proliferation was declined after addition of TGFß2 antibodies to co-incubated GBM cells with EZH2 inhibition. Besides, we found that EZH2 suppression in GBM cells enhanced co-culturing microglia engulfment through activation of iNOS. CONCLUSIONS: Our data demonstrates that EZH2 participates in GBM-induced immune deficient and EZH2 suppression in GBM can remodel microglia immune functions, which is beneficial for understanding GBM pathogenesis and suggests potential targets for therapeutic approaches.


Assuntos
Neoplasias Encefálicas/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Glioblastoma/patologia , Microglia/patologia , Microambiente Tumoral/fisiologia , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo
7.
Int J Neurosci ; 127(12): 1059-1064, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28332424

RESUMO

PURPOSE: To evaluate the neuroprotective effects of Dl-3-n-butylphthalide (NBP) on patients with radiation-induced brain injury, a hospital-based, clinical retrospective cohort study was conducted. PATIENTS AND METHODS: Data were collected on patients diagnosed with radiation-induced brain injury from January 2009 to January 2015 in Department of Neurology, Sun Yat-Sen Memorial Hospital. All patients enrolled have received cranial radiotherapy and were diagnosed with radiation-induced brain injury. Patients fulfilling certain eligibility criteria were recruited for analysis. The clinical therapeutic effects were observed and evaluated by LENT/SOMA scores before and one month after treatment in these two groups, respectively. RESULTS: The therapeutic effects of headache (total efficiency 75.76%), eurologic deficit (total efficiency 81.58%), cognitive functions (total efficiency 77.78%) and MRI results (total efficiency 74.29%) were better in the experimental group than those in the control group (p < 0.05). Nevertheless, there was no significant difference in mood and personality changes between these two groups. CONCLUSIONS: Administration of Dl-3-n-butylphthalide, in adjunct to corticosteroid therapy, might provide a better outcome in patients with radiation-induced brain injury.


Assuntos
Corticosteroides/farmacologia , Benzofuranos/farmacologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/fisiopatologia , Irradiação Craniana/efeitos adversos , Fármacos Neuroprotetores/farmacologia , Avaliação de Resultados em Cuidados de Saúde , Lesões por Radiação/tratamento farmacológico , Corticosteroides/administração & dosagem , Adulto , Benzofuranos/administração & dosagem , Lesões Encefálicas/etiologia , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Estudos Retrospectivos
8.
J Headache Pain ; 18(1): 86, 2017 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-28831756

RESUMO

OBJECTIVES: As a non-invasive therapy, whether transcranial magnetic stimulation (TMS) is effective on migraine. This article was aimed to assess the efficacy of TMS on migraine based on randomized controlled trails (RCTs). METHODS: We searched PubMed, Embase and Cochrane Library electronic databases for published studies which compared TMS group with sham group, conducted a meta-analysis of all RCTs. RESULTS: Five studies, consisting of 313 migraine patients, were identified. Single-pulse transcranial magnetic stimulation is effective for the acute treatment of migraine with aura after the first attack (p = 0.02). And, the efficacy of TMS on chronic migraine was not significant (OR 2.93; 95% CI 0.71-12.15; p = 0.14). CONCLUSIONS: TMS is effective for migraine based on the studies included in the article.


Assuntos
Transtornos de Enxaqueca/terapia , Manejo da Dor/métodos , Estimulação Magnética Transcraniana , Bases de Dados Factuais , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Estimulação Magnética Transcraniana/métodos
9.
Int J Neurosci ; 126(3): 269-77, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26000727

RESUMO

PURPOSE: The family of Toll-like receptors (TLRs) has recently been reported to play a role in ischemic injury, but the time course and cell types of the post-stroke TLR9 upregulation remain unclear. In this study, we investigated the dynamic changes of TLR9 expression and the expression of TLR9 in neurons and glial cells after cerebral ischemia reperfusion in mice. METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 90 min in male C57BL/6 mice. The TLR9 expression levels in the tissue surrounding the infarct were detected by Western Blot at 6 h, 3 d, 7 d, 14 d, 21 d, and 28 d after reperfusion. The expression of TLR9 in neurons and glial cells was observed by immunofluorescence staining. RESULTS: The expression of TLR9 protein first increased and then decreased, with the peak observed at 14 d-21 d. Only small punctate intracellular TLR9 was occasionally observed in the neurons at each time point, and the TLR9-positive rate showed no difference at different time points. By contrast, the activated microglia gathered at the margin of the infarct, and the intracellular TLR9 changed from scattered small punctate to coarse and lumpy. The TLR9-positive rate of microglia was first increased and then decreased with time, with the peak observed at 3 d. No positive TLR9 staining was found in the astrocytes and oligodendrocytes. CONCLUSIONS: TLR9 expression showed dynamic changes for a long period of time and microglias were the main brain cells to express TLR9 after cerebral ischemia and reperfusion.


Assuntos
Isquemia Encefálica/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Traumatismo por Reperfusão/metabolismo , Receptor Toll-Like 9/metabolismo , Regulação para Cima , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo
10.
J Transl Med ; 13: 359, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26576535

RESUMO

BACKGROUND: Autophagy is demonstrated to be involved in acute ischemic stroke(AIS), which, however, is confined to cells and/or animals levels. The aim of this study was to determine two autophagy biomarkers, Beclin1 and LC3B, in cerebrospinal fluid (CSF) and serum of patients with AIS, and to evaluate a possible correlation between levels of Beclin1 and LC3B and severity of neurological deficit and clinical outcome of stroke patients. METHODS: Levels of Beclin1 and LC3B were quantified by ELISA in CSF and serum collected from 37 AIS patients and 21 controls. The clinical severity at stroke onset was determined by the National Institute of Health Stroke Scale (NIHSS) and the neurological outcome was determined by the Modified Rankin Scale (mRs) and the improvement in NIHSS between stroke onset and 3 months later. Associations between autophagy biomarkers and infarct volume, NIHSS and mRs were assessed using Pearson analysis. RESULTS: The levels of Beclin1 and LC3B were increased both in CSF and serum of AIS patients relative to controls. In CSF, they were positively correlated with infarct volume and NIHSS scores, and negatively correlated with mRs scores, but no significant association was observed in serum. Moreover, AIS patients with higher levels of Beclin1 and LC3B in CSF had significantly higher improvement in NIHSS. CONCLUSION: CSF and serum levels of autophagy biomarkers are altered in AIS patients. CSF levels of autophagy biomarkers are associated with infarct volume, clinical severity of and neurological outcome.


Assuntos
Autofagia , Biomarcadores/líquido cefalorraquidiano , Isquemia Encefálica/líquido cefalorraquidiano , Acidente Vascular Cerebral/líquido cefalorraquidiano , Idoso , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/fisiopatologia
11.
Mol Cancer ; 13: 191, 2014 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-25128329

RESUMO

BACKGROUND: The successful gene delivery into the brain is a major challenge due to the presence of the blood-brain barrier (BBB). In order to transport plasmid DNA across the BBB and target the brain glioma, the PEGylated liposomes (PLs) modified with OX26 and chlorotoxin (CTX) were developed as a dual-targeting gene delivery system, and the therapeutic efficacy of OX26/CTX-PL/pC27 against glioma was evaluated using in vitro and in vivo experimental models. METHODS: The PEGylated liposome complexes were prepared by the reverse phase evaporation method, and their physicochemical properties were examined. The transfection efficiency, intracellular distribution, in vitro effects of OX26/CTX-PL/pC27 were determined on C6, F98 and HEK293T cell lines. The dual-targeting therapeutic efficacy of OX26/CTX-PL/pC27 against glioma were assessed using the BMVECs/C6 cells co-culture model and the rat orthotopic glioma model. RESULTS: The OX26/CTX-PL/pDNA complexes exhibited a subglobose shape, and possessed notably low toxicities to HEK293T and C6 cells post 4 h incubation. In the in vitro transfection experiment, gene expressions of hTERTC27 from C6 and F98 cells were significantly improved by OX26 and CTX modification. Our in vitro results also showed that OX26 endowed the PLs with the transport ability across the BBB. Using the BMVECs/C6 cells co-culture model, the viability of C6 cells was decreased to 46.0% after OX26/CTX-PL/pC27 transfection. The OX26/CTX-PL/pC27 complexes exhibited enhanced therapeutic effects on C6 cells. Moreover, the dual-targeting therapeutic effects were further conformed with diminished tumor volumes (18.81 ± 6.15 mm³) and extended median survival time (46 days) in C6 glioma-bearing rats. Immunohistochemical analysis revealed the therapeutic effects derived from enhanced hTERTC27 expression in the tumor site. CONCLUSIONS: The PEGylated liposomes modified with OX26 and CTX are able to significantly promote cell transfection, increase the transport of plasmid DNA across the BBB and afterwards target the brain glioma cells in vitro and in vivo, exhibit the most significant therapeutic efficacy. The ligand OX26 plays a critical role in transporting the lipoplexes across the BBB, and CTX acts as a major role in targeting brain glioma cells. The results would encourage further developments for non-invasive targeting therapy of brain gliomas by intravenous injection.


Assuntos
Anticorpos Monoclonais Murinos/metabolismo , Neoplasias Encefálicas/metabolismo , Técnicas de Transferência de Genes , Glioma/metabolismo , Lipossomos/metabolismo , Venenos de Escorpião/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Western Blotting , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Glioma/patologia , Células HEK293 , Humanos , Masculino , Tamanho da Partícula , Plasmídeos/metabolismo , Ratos Wistar , Eletricidade Estática , Transfecção
12.
Alcohol Clin Exp Res ; 38(10): 2529-40, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25346502

RESUMO

BACKGROUND: Alcohol consumption induces inflammatory damage in vessels, and the underlying mechanism is unclear. Valsartan, as one of the angiotensin receptor blockers (ARBs), plays a role in the inhibition of inflammatory reactions in vascular dysfunction. This study is to investigate the role of Toll-like receptor 2 (TLR2) in alcohol-induced inflammatory damage in vascular endothelial cells in vitro and to explore the protective effect of valsartan on alcohol-induced and TLR2-mediated inflammatory damage. METHODS: The human umbilical vein cell line (EA.hy926) were exposed to alcohol at 0 to 80 mM for 0 to 48 hours with or without valsartan pretreatment. The expression of TLR2 signaling, including TLR2, tumor necrosis factor receptor associated factor 6 (TRAF-6) and nuclear factor kappa B (NF-κB) p65 were detected by Western blot. The levels of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were determined by ELISA. To confirm the role of TLR2, we functionally up-regulated or down-regulated TLR2 by using TLR2 agonist or TLR2 small interfering RNA (siRNA), respectively. To further investigate the mechanism of alcohol on renin-angiotensin system, we detected the expression of angiotensin II receptor type 1 (AGTR1) in protein levels. RESULTS: The expression of TLR2, TRAF-6, NF-κB p65, and the proinflammatory cytokines, TNF-α and IL-6, were significantly increased after alcohol exposure in EA.hy926 endothelial cells. This was enhanced by TLR2 agonist, and was inhibited by TLR2 siRNA transfection. The pretreatment of valsartan resulted in an inhibition of TLR2 signaling and proinflammatory cytokines. The expression of AGTR1 was up-regulated after alcohol exposure, and was blocked by valsartan pretreatment. CONCLUSIONS: TLR2 signaling-mediated alcohol induced inflammatory response in human vascular epithelial cells in vitro, which was inhibited by valsartan.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Etanol/efeitos adversos , Inflamação/prevenção & controle , Inflamação/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Tetrazóis/farmacologia , Receptor 2 Toll-Like/efeitos dos fármacos , Valina/análogos & derivados , Antagonistas de Receptores de Angiotensina/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Etanol/farmacologia , Humanos , Técnicas In Vitro , Inflamação/induzido quimicamente , Interleucina-6/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 2 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Valina/farmacologia , Valsartana
13.
J Stroke Cerebrovasc Dis ; 23(10): 2671-2680, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25307427

RESUMO

BACKGROUND: The effect of changing temperature on an individual's cerebrovascular risk is both biologically plausible and supported by epidemiologic evidence. We used a global proteomic-based approach to analyze the expression alterations of proteins in artificial cold exposure (ACE)-induced hypertensive stroke in renovascular hypertensive rats (RHR) and to identify the biomarker of ACE-induced hypertensive stroke. METHODS: The RHR models were established by 2 kidney 2 clip methods. ACE treatment was achieved using an intelligent artificial climate cabinet. Blood pressure and neurologic symptoms were observed before and after ACE treatment. Hemorrhagic condition and infarction survey were examined using 2,3,5-triphenyltetrazolium chloride staining. The total number of proteins derived from the cerebral tissue of the RHR models were analyzed with 2-dimensional gel electrophoresis (2-DE), ImageMaster 2D Platinum software, and mass spectrometry. Significantly regulated proteins selected for further functional studies using the Search Tool for the Retrieval of Interacting Genes/Proteins system were verified by Western blot. RESULTS: ACE-induced stroke in the RHR group (31.25%, 25 of 80 vs. 16.25%, 13 of 80; P < .05) but not in the sham-operated group. Following ACE treatment, we identified 37 differentially expressed proteins and 28 were unique. Two of the upregulated proteins, Syt1 and Idh3a, were obtained by bioinformatics analysis and verified by Western blot. CONCLUSIONS: The rate of morbidity as a result of stroke in RHR was obviously elevated after ACE treatment. ACE might affect protein expression profile in cerebral tissues of RHR. Syt1 and Idh3a may play a vital role in ACE-induced hypertensive stroke.


Assuntos
Biomarcadores/metabolismo , Temperatura Baixa/efeitos adversos , Hipertensão Renal/complicações , Rim/irrigação sanguínea , Circulação Renal , Acidente Vascular Cerebral/metabolismo , Animais , Pressão Sanguínea , Western Blotting , Modelos Animais de Doenças , Isocitrato Desidrogenase/metabolismo , Rim/cirurgia , Masculino , Proteômica , Ratos , Acidente Vascular Cerebral/etiologia , Sinaptotagmina I/metabolismo , Sais de Tetrazólio/química
14.
CNS Neurosci Ther ; 30(3): e14689, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38516831

RESUMO

AIMS: Chronic alcohol exposure leads to persistent neurological disorders, which are mainly attributed to neuroinflammation and apoptosis. Stimulator of IFN genes (STING) is essential in the cytosolic DNA sensing pathway and is involved in inflammation and cellular death processes. This study was to examine the expression pattern and biological functions of STING signaling in alcohol use disorder (AUD). METHODS: Cell-free DNA was extracted from human and mouse plasma. C57BL/6J mice were given alcohol by gavage for 28 days, and behavior tests were used to determine their mood and cognition. Cultured cells were treated with ethanol for 24 hours. The STING agonist DMXAA, STING inhibitor C-176, and STING-siRNA were used to intervene the STING. qPCR, western blot, and immunofluorescence staining were used to assess STING signaling, inflammation, and apoptosis. RESULTS: Circulating cell-free mitochondrial DNA (mtDNA) was increased in individuals with AUD and mice chronically exposed to alcohol. Upregulation of STING signaling under alcohol exposure led to inflammatory responses in BV2 cells and mitochondrial apoptosis in PC12 cells. DMXAA exacerbated alcohol-induced cognitive impairment and increased the activation of microglia, neuroinflammation, and apoptosis in the medial prefrontal cortex (mPFC), while C-176 exerted neuroprotection. CONCLUSION: Activation of STING signaling played an essential role in alcohol-induced inflammation and mitochondrial apoptosis in the mPFC. This study identifies STING as a promising therapeutic target for AUD.


Assuntos
Disfunção Cognitiva , Doenças Neuroinflamatórias , Humanos , Camundongos , Animais , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Inflamação/induzido quimicamente , Inflamação/metabolismo , Etanol/toxicidade , DNA Mitocondrial/metabolismo , Apoptose , Disfunção Cognitiva/induzido quimicamente
15.
Free Radic Biol Med ; 220: 15-27, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38679301

RESUMO

BACKGROUND: Chronic alcohol exposure induces cognitive impairment and NLRP3 inflammasome activation in the mPFC (medial prefrontal cortex). Mitophagy plays a crucial role in neuroinflammation, and dysregulated mitophagy is associated with behavioral deficits. However, the potential relationships among mitophagy, inflammation, and cognitive impairment in the context of alcohol exposure have not yet been studied. NRF2 promotes the process of mitophagy, while alcohol inhibits NRF2 expression. Whether NRF2 activation can ameliorate defective mitophagy and neuroinflammation in the presence of alcohol remains unknown. METHODS: BV2 cells and primary microglia were treated with alcohol. C57BL/6J mice were repeatedly administered alcohol intragastrically. BNIP3-siRNA, PINK1-siRNA, CCCP and bafilomycin A1 were used to regulate mitophagy in BV2 cells. RTA-408 acted as an NRF2 activator. Mitochondrial dysfunction, mitophagy and NLRP3 inflammasome activation were assayed. Behavioral tests were used to assess cognition. RESULTS: Chronic alcohol exposure impaired the initiation of both receptor-mediated mitophagy and PINK1-mediated mitophagy in the mPFC and in vitro microglial cells. Silencing BNIP3 or PINK1 induced mitochondrial dysfunction and aggravated alcohol-induced NLRP3 inflammasome activation in BV2 cells. In addition, alcohol exposure inhibited the NRF2 expression both in vivo and in vitro. NRF2 activation by RTA-408 ameliorated NLRP3 inflammasome activation and mitophagy downregulation in microglia, ultimately improving cognitive impairment in the presence of alcohol. CONCLUSION: Chronic alcohol exposure-induced impaired mitophagy initiation contributed to NLRP3 inflammasome activation and cognitive deficits, which could be alleviated by NRF2 activation via RTA-408.


Assuntos
Disfunção Cognitiva , Inflamassomos , Proteínas de Membrana , Microglia , Mitofagia , Fator 2 Relacionado a NF-E2 , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Mitofagia/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Inflamassomos/metabolismo , Inflamassomos/genética , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/patologia , Microglia/metabolismo , Microglia/efeitos dos fármacos , Microglia/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Etanol/toxicidade , Etanol/efeitos adversos
16.
J Stroke Cerebrovasc Dis ; 22(8): e477-85, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23800500

RESUMO

Our objectives are to investigate the role of MMP-9 in cold exposure-induced stroke and assess the preventive effect of doxycycline, a total of 200 rats were assigned to a control group, sham group, 2-kidney, 2-clip (2K-2C) group, and doxycycline-received 2K-2C group (2K-2C+doxy) (N=50, each), and subsequently, each group were randomly assigned to 2 groups: acute cold exposure (ACE) and nonacute cold exposure (NACE) (N=25, each). After the blood pressure was stabilized, rats were maintained on a 12-h light (22°C)/dark (4°C) cycle (ACE group) or a 12-h light (22°C)/dark (22°C) cycle (NACE group) for 3 cycles. The results showed that ACE upregulated Ang II and MMP-9 protein levels in brains and aortas and considerably enhanced stroke incidence in 2K-2C rats. In contrast, doxycycline treatment prevented upregulation of MMP-9 protein expression and activity in brains and aortas in response to ACE and significantly decreased stroke incidence. These findings suggest that cold exposure-induced MMP-9 via activation of RAS might play a critical role in the initiation of cold exposure-induced stroke during chronic hypertension and doxycycline shows protective effects against cold exposure-induced stroke.


Assuntos
Aorta/enzimologia , Encéfalo/enzimologia , Hipertensão Renovascular/complicações , Hipotermia Induzida/efeitos adversos , Metaloproteinase 9 da Matriz/metabolismo , Acidente Vascular Cerebral/etiologia , Angiotensina II/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Pressão Sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Doxiciclina/farmacologia , Hipertensão Renovascular/fisiopatologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Sistema Renina-Angiotensina , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/prevenção & controle , Fatores de Tempo
17.
Mol Neurobiol ; 60(5): 2749-2766, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36717480

RESUMO

The pyrin domain-containing protein 3 (NLRP3) inflammasome drives the profound cerebral ischemia and reperfusion injury (I/R) and mediates the secretion of IL-1ß (interleukin-1ß), which exerts a subsequent cascade of inflammatory injury. The NLRP3-activated-microglial manipulation in adjacent neuronal and endothelial NLRP3 activation has been confirmed in our previous studies. In the present study, we extended the cognition of how microglia mediated neuronal and endothelial NLRP3-IL-1ß signaling during cerebral ischemia and reperfusion injury. In vitro, Neuro-2a and bEND3 cells were cultured alone or co-cultured with BV2 cells and oxygen-glucose deprivation/reoxygenation (OGD/R) was performed. In vivo, transient middle cerebral artery occlusion (tMCAO) rat models and lentiviral silencing targeting IL-1R1 were performed. The NLRP3 inflammasome activation was evaluated by enzyme-linked immunosorbent assay, western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence. In the co-culture system after OGD/R treatment, NLRP3 inflammasomes in neurons and endothelial cells were activated by microglial IL-1ß via IL-1ß/IL-1R1/TRAF6 signaling pathway, with the basal protein level of NLRP3. In addition, ruptured lysosomes engulfing ASC specks which were possibly secreted from microglia triggered the enhanced NLRP3 expression. In cortices of tMCAO rats at 24 h of reperfusion, silencing IL-1R1, mainly presented in neurons and endothelial cells, was efficient to block the subsequent inflammatory damage and leukocyte brain infiltration, leading to better neurological outcome. Neuronal and endothelial NLRP3 inflammasomes were activated by microglia in cerebral ischemia and reperfusion injury mainly via IL-1ß/IL-1R1/TRAF6 signaling, which might be therapeutically targetable.


Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Animais , Ratos , Isquemia Encefálica/metabolismo , Células Endoteliais/metabolismo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Traumatismo por Reperfusão/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo
18.
J Psychiatr Res ; 164: 150-161, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37352811

RESUMO

Morphine is one of the most effective and widely used analgesic drugs. However, chronic morphine use caused opioid-induced hyperalgesia (OIH). The development of OIH limits the use of morphine. The mechanisms of OIH are not fully understood. Toll-like receptor4 (TLR4) and glutamate receptors in the periaqueductal gray (PAG) are critical in OIH, however, the association between TLR4 and N-methyl-D-aspartate Receptors (NMDARs) activation in PAG remains unclear. Microglia activation, increased TLR4/p65 nuclear factor-kappa B (p65 NF-κB) and proinflammatory cytokines in microglia, and phosphorylation of NMDAR1 subunit (NR1) and NMDAR2B subunit (NR2B) in neurons were observed in PAG of OIH mice. Up-regulations of TLR4/p65 NF-κB and proinflammatory cytokines (IL-1ß, IL-6, TNF-α) in BV2 cells were prevented by inhibiting and knocking down TLR4. By inhibiting myeloid differentiation factor 2 (MD2) and knocking down the High-mobility group box 1 (HMGB1), we found that morphine activated TLR4 by HMGB1 but not MD2. We co-cultured Neuro-2a (N2A) with BV2 microglial cell line and found that instead of directly phosphorylating NMDAR subunits, morphine increased the phosphorylation of NR1 and NR2B by inducing TLR4-mediated microglia inflammation. Knocking TLR4 out of PAG by Lentivirus-GFP-TLR4 shRNA reversed these changes and relieved OIH. Our findings suggested that the secretion of HMGB1 induced by morphine-activated TLR4 in microglia, and the proinflammatory factors released by activated microglia phosphorylated NR1 and NR2B of adjacent neurons, induced increased neuronal excitability. In conclusion, TLR4/NMDARs in PAG were involved in the development and maintenance of OIH and supported novel strategies for OIH treatment.


Assuntos
Proteína HMGB1 , Morfina , Camundongos , Animais , Morfina/efeitos adversos , Morfina/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , NF-kappa B/metabolismo , Microglia/metabolismo , Receptor 4 Toll-Like/metabolismo , Substância Cinzenta Periaquedutal/metabolismo , Transdução de Sinais , Proteína HMGB1/efeitos adversos , Proteína HMGB1/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Analgésicos Opioides/efeitos adversos , Citocinas/metabolismo , Neurônios
19.
Exp Neurol ; 353: 114061, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35367455

RESUMO

BACKGROUND AND AIMS: Long-term alcohol intake leads to cognitive impairment and dementia. The impairment of the cerebral cortex and limbic structures in alcoholics is associated with the loss of synapses instead of neurons. Synapse loss is considered to be an early and key feature of many neurodegenerative diseases, in which microglia-mediated synapse elimination is vital. However, the underlying mechanisms of synapse loss and cognitive impairment caused by long-term alcohol intake are still largely unknown. METHODS: We investigated the relationship of synapse impairment, the microglial innate immune receptor-TREM2, and microglia-mediated synaptic elimination in long-term alcohol exposure. RESULTS: We found that long-term alcohol exposure increased expression of TREM2, decreased expression of synaptic proteins and glutamate receptor subunits, reduced dendrite spine density, and impaired long-term potentiation (LTP) in the hippocampus. Minocycline reduced the amount of the postsynaptic marker PSD95 in microglia, attenuated dendrite spine density loss, and slow down the forgetting process of already-formed memory. Furthermore, we found that TREM2 participated in microglia-mediated synapse elimination in chronic alcohol exposure in vivo. Significantly fewer PSD95 were detectable in microglial phagolysosomes in TREM2 knockdown mice. Besides, TREM2 gene silencing ameliorated synapse loss, LTP impairment, and forgetting of remote memories. CONCLUSIONS: Our data suggests that TREM2 is associated with synaptic plasticity impairment and memory deficits, indicating microglia-mediated synaptic pruning might be the underlying mechanism involved in synapse loss and memory impairment induced by long-term alcohol intake. These findings provide new evidence for the receptor's participation in neurodegeneration diseases.


Assuntos
Disfunção Cognitiva , Microglia , Animais , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/metabolismo , Hipocampo/metabolismo , Glicoproteínas de Membrana/metabolismo , Transtornos da Memória/metabolismo , Camundongos , Camundongos Knockout , Microglia/metabolismo , Plasticidade Neuronal/fisiologia , Receptores Imunológicos/metabolismo , Sinapses/metabolismo
20.
Addiction ; 117(5): 1382-1391, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34859517

RESUMO

BACKGROUND AND AIMS: Observational studies have yielded conflicting results on the association of prescription opioid use (POU) with the risk of cardiovascular diseases (CVD). Residual confounding and potential reverse causality are inevitable in such conventional observational studies. This study used Mendelian randomization (MR) design to estimate the causal effect of POU on the risk of CVD, including coronary heart disease (CHD), myocardial infarction (MI) and ischemic stroke (IS), as well as their common risk factors. DESIGN: We estimated the causal effect of genetic liability for POU on CVD in a two-sample MR framework. Complementary sensitivity analyses were conducted to test the robustness of our results. SETTING: Genome-wide association studies (GWAS) that were based on predominantly European ancestry. PARTICIPANTS: The sample sizes of the GWAS used in this study ranged from 69 033 to 757 601 participants. MEASUREMENTS: Genetic variants predictive of the POU and their corresponding summary-level information in the outcomes were retrieved and extracted from the respective GWAS. FINDINGS: Using univariable MR, we found evidence for a causal effect of genetic liability for POU on an increased risk of CHD [odds ratio (OR) = 1.09; 95% confidence interval (CI) = 1.02-1.16; P = 0.008] and MI (OR = 1.13; 95% CI = 1.04-1.22; P = 0.002). In multivariable MR, the association remained after accounting for comorbid pain conditions, but was attenuated with adjustment for potential mediators, including body mass index (BMI), waist circumference (WC) and type 2 diabetes (T2D). CONCLUSION: Mendelian randomization estimates provide robust evidence for the causal effects of genetic liability for prescription opioid use on an increased risk of coronary heart disease and myocardial infarction, which might be mediated by obesity-related traits.


Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Analgésicos Opioides/efeitos adversos , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Estudo de Associação Genômica Ampla , Humanos , Análise da Randomização Mendeliana , Polimorfismo de Nucleotídeo Único , Prescrições , Fatores de Risco
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